Research Paper Ursolic acid reverses liver fibrosis by …...Ursolic acid reverses liver fibrosis by inhibiting interactive NOX4/ROS and RhoA/ROCK1 signalling pathways Sizhe Wan1,*,

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INTRODUCTION

Hepatic fibrosis is the net accumulation of extracellular

matrix (ECM) resulting from chronic liver injury of any

aetiology, including viral infection, alcohol

consumption, non-alcoholic fatty liver disease,

cholestasis, and autoimmune liver disease [1, 2]. ECM

accumulation then induces fibrous connective tissue

hyperplasia, replacing the space in which normal

hepatocyte regeneration occurs [3]. Sustained hepatic

fibrosis can lead to cirrhosis, which contributes to more

than 1 million deaths per year worldwide [4, 5]; despite

this high mortality rate, there is currently no approved

anti-fibrotic treatment. Hepatic stellate cells (HSCs) are

activated by injury and release ECM, the deposition of

which is a central event of liver fibrosis [6]. Once

chronic liver disease progresses to end-stage liver

disease, there are no effective treatments other than liver

transplantation, which is limited by donor shortages,

high costs, and immune rejection. Therefore, the

reversibility of liver fibrosis has been the subject of

extensive research.

NADPH oxidase (NOX) is a multi-subunit trans-

membrane enzyme complex composed of seven

members: NOX1, NOX2, NOX3, NOX4, NOX5 and the

two dual oxidases Duox1 and Doux2. The subunits of

NOX are slightly different and participate in liver fibrosis

by generating reactive oxygen species (ROS) to regulate

HSC signal transduction [7]. NOX4, an important subtype

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Research Paper

Ursolic acid reverses liver fibrosis by inhibiting interactive NOX4/ROS and RhoA/ROCK1 signalling pathways

Sizhe Wan1,*, Fangyun Luo1,*, Chenkai Huang1, Cong Liu1, Qingtian Luo1, Xuan Zhu1 1Department of Gastroenterology, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China *Equal contribution

Correspondence to: Xuan Zhu; email: [email protected] Keywords: ursolic acid,l iver fibrosis, NOX4, RhoA, ROCK1 Received: February 13, 2020 Accepted: April 20, 2020 Published: June 3, 2020

Copyright: Wan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

ABSTRACT

Liver fibrosis is the reversible deposition of extracellular matrix (ECM) and scar formation after liver damage by various stimuli. The interaction between NOX4/ROS and RhoA/ROCK1 in liver fibrosis is not yet clear. Ursolic acid (UA) is a traditional Chinese medicine with anti-fibrotic effects, but the molecular mechanism underlying these effects is still unclear. We investigated the interaction between NOX4/ROS and RhoA/ROCK1 during liver fibrosis and whether these molecules are targets for the anti-fibrotic effects of UA. First, we confirmed that UA reversed CCl4-induced liver fibrosis. In the NOX4 intervention and RhoA intervention groups, related experimental analyses confirmed the decrease in CCl4-induced liver fibrosis. Next, we determined that the expression of NOX4 and RhoA/ROCK1 was decreased in UA-treated liver fibrotic mice. Furthermore, RhoA/ROCK1 expression was decreased in the NOX4 intervention group, but there was no significant change in the expression of NOX4 in the RhoA intervention group. Finally, we found that liver fibrotic mice showed a decline in their microbiota diversity and abundance, a change in their microbiota composition, and a reduction in the number of potential beneficial bacteria. However, in UA-treated liver fibrotic mice, the microbiota dysbiosis was ameliorated. In conclusion, the NOX4/ROS and RhoA/ROCK1 signalling pathways are closely linked to the development of liver fibrosis. UA can reverse liver fibrosis by inhibiting the NOX4/ROS and RhoA/ROCK1 signalling pathways, which may interact with each other.

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of the NOX family, has been shown to induce the

conversion of HSCs to myofibroblasts (MFBs) by

releasing ROS, which is closely related to liver fibrosis.

This function indicates that NOX4/ROS play an important

role in the development of liver fibrosis.

To date, more than 20 Rho family members have been

discovered. The RhoA subfamily is a group of small

GTPase proteins that belong to the Rho protein family,

which in turn belongs to the Ras superfamily; when

activated, these small proteins act as molecular switches

to regulate the cyclical transformation between the

activated GTP-binding state and the inactivated GDP-

binding state. RhoA binds to multiple target proteins,

including epidermal growth factor receptor (EGFR) and

Rho-associated coiled-coil-forming protein kinase

(ROCK), and regulates cytoskeletal dynamics and gene

transcription [8], thereby regulating the adhesion,

movement, and contraction of HSCs and participating in

the development of liver fibrosis [9].

Studies have shown that Rho GTPases, especially Rac1,

can regulate the activation of NOX1 and NOX2 [10],

indicating a link between the Rho GTPase family and

the NOX family. However, there is controversy about

the relationship between NOX4/ROS and RhoA/ROCK.

Recent studies have indicated that in pulmonary fibrosis,

NOX4/ROS can activate the RhoA/ROCK signalling

pathway, promote lung fibroblast migration and collagen

synthesis, and enhance pulmonary fibrosis development

[11]. However, the role of NOX4/ROS in kidney fibrosis

is different from that in pulmonary fibrosis.

RhoA/ROCK are upstream signalling molecules of

NOX4/ROS. Activation of the RhoA/ROCK signalling

pathway can upregulate NOX4/ROS expression,

promote renal muscle fibroblast differentiation, and

aggravate renal fibrosis [12]. The mechanism of

interaction between NOX4/ROS and RhoA/ROCK in

liver fibrosis has not been determined, although both

NOX4/ROS and RhoA/ROCK are involved in regulating

HSC activation in association with the progression of

fibrotic disease [11, 13].

Ursolic acid (UA), a traditional Chinese medicine, is a

natural pentacyclic triterpenoid compound derived from

Chinese medicine plants and has been reported to have

anti-inflammatory, anti-fibrotic, and liver protective

properties [14, 15]. Although previous studies have

confirmed that UA could reverse liver fibrosis by

inhibiting the activation of HSCs and promoting

apoptosis [16], the specific mechanism of action

involved is not clear. This study mainly explored the

potential mechanism of the anti-fibrotic properties of

UA, providing powerful experimental support for the

future clinical application of UA in the treatment of

patients with liver fibrosis.

RESULTS

UA reverses liver damage and fibrosis in liver

fibrotic mice

First, we used HE and Masson’s trichrome staining

of mouse liver sections to determine the effects of UA

on liver damage, fibrous septum formation and

collagen deposition in mice with liver fibrosis (Figure

1A–1B). After CCl4-induced mice were treated

with UA, the effects of CCl4 on liver hepatic lobule

structure, collagen deposition and fibrous connective

tissue hyperplasia were significantly reversed and

were accompanied by decreased inflammatory cell

infiltration (P<0.05) (Figure 1C–1D). Furthermore, as

an important component of collagen deposition during

liver fibrosis, the hydroxyproline content in the liver

tissue was also detected (Figure 1E). Next, we

assessed the levels of ALT, AST, and TBIL in the

mouse serum to determine liver function (Figure 1F).

Compared to those in the control group, the

serum levels of ALT, AST and TBIL in the CCl4

group mice were significantly increased. However,

this increase was inhibited in UA-treated fibrotic

mice. These results indicate that UA can reverse liver

damage and fibrosis to a certain extent to protect

the liver.

HSC activation is well established as the central driver of

hepatic fibrosis in experimental and human liver injury,

and HSC activation releases a large amount of ECM,

which is deposited in the interstitial space of the liver,

eventually leading to liver fibrosis [19]. As shown by

both α-SMA staining and the TUNEL assay, the

expression of α-SMA, a biomarker of activated HSCs

[20], in the liver tissue of the CCl4 group was

significantly increased compared to that in the control

group. Apoptosis in hepatocytes in the CCl4 group also

increased, but there was a significant decrease in

apoptosis in the UA group (Figure 2A). This indicates

that UA can inhibit the apoptosis of hepatocytes and the

activation of HSCs, thereby inhibiting the progression of

liver fibrosis.

Liver fibrosis is often accompanied by changes in the

expression of fibrosis-related factors. At the mRNA

and protein levels, the expression levels of type I

collagen and TIMP-1 in the CCl4 group were

significantly elevated compared with those in the

control group. After UA treatment, this increase in

the expression of type I collagen and TIMP-1

decreased, and the expression of MMP-1, an anti-

fibrotic factor that promotes the degradation of

extracellular matrix [21], was elevated (Figure 2B–

2C), demonstrating that UV exerts a reversal effect

on liver fibrosis.


UA relieves liver fibrosis by inhibiting the

NOX4/ROS signalling pathway

NOX4 mediates the signal transduction of major pro-

hepatic fibrosis factors, such as TGF-β, leading to HSC

activation and hepatocyte apoptosis and thus plays an

important role in liver fibrosis [22]. Our previous

studies confirmed that NOX4 and ROS are upregulated

in fibrotic liver tissue and that Rac1 expression is

upregulated in activated HSCs. In CCl4-induced liver

fibrotic mice, the NOX4 expression level MDA content

that assessed the accumulation of ROS and oxidative

stress in the liver were significantly increased (Figure

3A–3D). To further validate the importance of NOX4 in

liver fibrosis, we generated NOX4-/- liver fibrotic mice

and injected liver fibrotic mice with the NOX4 inhibitor

AP (Supplementary Figure 1). Compared with the mice

in the CCl4 group, the mice in the NOX4-/- and AP

groups had a smaller fibrous septum and less collagen

deposition, which were accompanied by decreased

inflammatory cell infiltration (Figure 1A–1E). The

levels of serum markers suggested that liver function

damage was reduced in liver fibrotic mice after NOX4

inhibition (Figure 1F). The results of other experiments,

such as α-SMA staining, TUNEL assay, MDA content,

and liver fibrosis-related factor expression analyses,

were similar (Figure 2A–2C), indicating that the

inhibition of NOX4 can improve liver fibrosis,

confirming that NOX4 is an important profibrotic factor

in the progression of liver fibrosis.

To confirm that NOX4 is a target for UA intervention in

fibrosis, we first verified the effect of UA on NOX4

expression in vivo by IHC (Figure 3A). The expression

of NOX4 in the UA group was lower than that in the

CCl4 group. Changes in the mRNA expression of

NOX4 also confirmed this hypothesis (Figure 3B). The

mRNA expression level of NOX4 was significantly

lower in the UA group than in the CCl4 group (P<0.01).

Changes in NOX4 expression at the protein level were

also similar to those at the mRNA level (Figure 3C).

Moreover, the MDA content in the UA group was lower

than that in the CCl4 group (Figure 3D), indicating the

inhibitory effect of UA on NOX4/ROS. Next, we

explored whether NOX4 is a target for the anti-fibrotic

effects of UA. In the NOX4-/-+UA group, changes to the

Figure 1. The effect of UA on CCl4-induced liver injury and fibrosis is related to NOX4. (A) HE staining (100× magnification). (B) Masson’s trichrome staining (100× magnification). (C–D) Morphometrical analysis of the fibrotic score and fibrotic area. (E) Detection of the hydroxyproline content in the liver tissue by colorimetry. (F) Liver function indices in mouse sera. Data represent the mean ± SD for each group. *P < 0.05 and ***P < 0.001.


Figure 2. The effect of UA on CCl4-induced liver fibrosis-related indicators is related to NOX4. (A) Dual immunofluorescence staining of liver sections from mice in the control, CCl4 and UA groups for nuclei (DAPI, blue), aHSCs (α-SMA, green), and apoptosis (TUNEL, red), and the merged images are shown. (B) Hepatic mRNA levels of collagen I, MMP-1, α-SMA, and TIMP-1 were measured by qRT-PCR. (C) Collagen I, MMP-1, α-SMA, and TIMP-1 protein expression was detected by a western blot. Data represent the mean ± SD of each group. *P < 0.05 and ***P < 0.001.


fibrous septum, collagen deposition and inflammatory

infiltration and serum indices were not more decreased

than those in the NOX4-/- group, although these changes

were still less pronounced than those in the CCl4 group

(Figure 1A–1F). Furthermore, compared to those in the

NOX4-/- group, the number and area of α-SMA- and

TUNEL-positive cells in the NOX4-/-+UA group were

not significantly changed. The changes in factors related

to liver fibrosis were similar (Figure 2A–2C),

suggesting that UA reverses liver fibrosis by inhibiting

the NOX4/ROS pathway.

RhoA/ROCK1 is another target of the anti-fibrotic

effect of UA

RhoA has been shown to regulate HSC activation,

migration, adhesion, contraction, proliferation and

apoptosis [9, 23, 24]. Compared with their expression

in the control group, RhoA and ROCK1 expression in

the CCl4 group was not significantly altered (Figure

4A–4C). To examine the effects of RhoA on liver

fibrosis in vivo, we constructed RhoA-inhibited liver

fibrotic mice by injecting the mice with AAV and the

RhoA inhibitor fasudil (Supplementary Figure 2).

Compared with mice in the CCl4 group, liver fibrotic

mice in the RhoAi and FA groups exhibited a smaller

fibrous septum, less collagen deposition and fewer

infiltrated inflammatory cells (Figure 5A–5D).

Correspondingly, the hydroxyproline content was

lower in RhoA-inhibited mice with liver fibrosis than

in control mice (Figure 5E). We next tested the levels

of ALT, AST, and TBIL in mouse serum. The liver

function in liver fibrotic mice in the RhoAi and FA

groups exhibited significant recovery, unlike that in

the CCl4 group (Figure 5F). The results of dual

immunofluorescence and the expression of liver

fibrosis-related factors also show the importance of

RhoA in liver fibrosis (Figure 6A–6C).

Figure 3. Effect of UA on the expression of NOX4 in mice with liver fibrosis. (A) The effect of UA on NOX4 expression was determined by using IHC. (B) Hepatic mRNA levels of NOX4 were measured by qRT-PCR. (C) Hepatic protein levels of NOX4 were detected by a western blot. (D) Detection of MDA content in the liver by a commercial kit. Data represent the mean ± SD of each group. *P < 0.05 and ***P < 0.001.


Figure 4. Effect of UA on the expression of RhoA/ROCK1 in liver fibrotic mice. (A) The effect of UA on RhoA/ROCK1 expression was determined by using IHC. (B) Hepatic mRNA levels of RhoA/ROCK1 were measured by qRT-PCR. (C) Hepatic protein levels of RhoA/ ROCK1 were detected by a western blot. Data represent the mean ± SD of each group. *P < 0.05 and ***P < 0.001.


We assessed whether UA reverses liver fibrosis by

inhibiting RhoA-related signalling pathways. First, to

verify the effect of UA on the expression of RhoA and

ROCK1, the downstream target of RhoA, we

conducted IHC analyses (Figure 4A). The expression

of RhoA/ROCK1 in the UA group was lower than that

in the CCl4 group. Changes in the mRNA and protein

levels of RhoA and ROCK1 were similar. In liver

fibrotic mice, UA significantly reduced this increase

in the expression of RhoA/ROCK1 in the liver

(P<0.05) (Figure 4B–4C), indicating that UA can

interfere with the expression of RhoA/ROCK. Next,

we explored whether the anti-fibrotic effect of UA is

related to its inhibition of RhoA/ROCK. Changes to

the fibrous septum, collagen deposition and

inflammatory infiltration in the RhoAi+UA group

were not significant compared with those in the

RhoAi group, although they were still less than those

in the CCl4 group (Figure 5A–5E). The levels of

serum indicators also recovered to a certain degree

compared with those in the RhoAi treatment group

(Figure 5F). Compared to those in the RhoAi group,

the number and area of α-SMA- and TUNEL-positive

cells were decreased in the RhoAi+UA group (Figure

6A). The expression of liver fibrosis-associated

factors also decreased in UA-treated liver fibrotic

mice following RhoA inhibition (Figure 6B–6C).

These results indicate that UA exerts anti-fibrotic

effects by inhibiting the RhoA/ROCK1 signalling

pathway.

NOX4/ROS is the upstream signalling pathway of

RhoA/ROCK1 in liver fibrosis

The above results confirm that NOX4/ROS and

RhoA/ROCK are two signalling pathways on which

Figure 5. The effect of UA on CCl4-induced liver injury and fibrosis is related to RhoA. (A) HE staining (100× magnification). (B) Masson’s trichrome staining (100× magnification). (C–D) Morphometrical analysis of the fibrotic score and fibrotic area. (E) Detection of the hydroxyproline content in liver tissue by colorimetry. (F) Liver function indices in mouse sera. Data represent the mean ± SD of each group. *P < 0.05 and ***P < 0.001.


Figure 6. The effect of UA on CCl4-induced liver fibrosis-related indicators is related to RhoA. (A) Dual immunofluorescence staining of liver sections from mice in the control, CCl4, and UA groups stained for nuclei (DAPI, blue), aHSCs (α-SMA, green), and apoptosis (TUNEL, red), and the merged images are shown. (B) Hepatic mRNA levels of collagen I, MMP-1, α-SMA, and TIMP-1 were measured by qRT-PCR. (C) Collagen I, MMP-1, and TIMP-1 protein expression was detected by a western blot. Data represent the mean ± SD of each group. *P < 0.05 and ***P < 0.001.


UA exerts its anti-fibrotic effects. We then sought to

clarify the possible interaction mechanism between

these two signalling pathways. First, we investigated

the expression of RhoA/ROCK1 in NOX4-inhibited

liver fibrotic mice. IHC analysis showed that the

expression of RhoA/ROCK1 in the NOX4-/- group was

decreased compared with that in the CCl4 group

(Figure 7A). Similarly, dual immunofluorescence

staining for α-SMA and RhoA showed decreased

expression of RhoA in NOX4-/- mice (Figure 7B). At

the mRNA level, the expression of RhoA/ROCK1 in

the NOX4-/- group was significantly decreased

compared to that in the CCl4 group (P<0.01) (Figure

7C). Next, we determined the expression of NOX4 in

RhoA-inhibited liver fibrotic mice. As shown by IHC,

the expression of NOX4 in the RhoAi group was not

significantly lower than that in the CCl4 group (Figure

7D). A slight decrease in the MDA content in the

livers of RhoA-inhibited liver fibrotic mice was

observed (Figure 7E). Compared to that in the CCl4

group, the mRNA level of NOX4 in the RhoAi group

was decreased to a certain degree, but this difference

was not statistically significant (Figure 7F), indicating

that NOX4/ROS is the upstream signalling pathway of

RhoA/ROCK1 in liver fibrosis.

Intestinal improvement in liver fibrotic mice

following treatment with UA

Intestinal damage and destruction of intestinal barrier

integrity are often accompanied by liver fibrosis [25–27].

Once the integrity of the intestine is destroyed,

harmful factors in the intestine enter the liver through

the gut-liver axis, which in turn further aggravates the

progression of liver fibrosis [28, 29]. Therefore, we

explored improvements to the intestinal barrier

induced by UA. As they are key indicators of the

integrity of the intestinal barrier, the expression of the

tight junction (TJ) proteins ZO-1 and occludin was

tested. The expression of ZO-1 and occludin in the

ileum of liver fibrotic mice was lower than that in the

control group, but their expression increased after UA

treatment (P<0.05) (Figure 8A). These results show

that UA has a protective effect on the intestinal

barrier.

The intestinal microbiota has also received attention as

an important indicator of intestinal and systemic status.

We explored disorders in the intestinal microbiota of

animal models of liver fibrosis and their improvement

following treatment with UA. Disorders in the

intestinal microbiota in our animal models of liver

fibrosis and improvements to the intestinal microbiota

following UA treatment were detected by next-

generation sequencing. First, we tested some indicators

that represent alpha diversity (Figure 8B). The Shannon

index and the Chao1 index, which were used to assess

microbial abundance, in the CCl4 group were

significantly decreased compared to those in the control

group (P<0.01). After UA treatment, these indices

increased (P<0.01). In the NOX4-/-+UA and

RhoAi+UA groups, this recovery in the Shannon and

Chao1 indices was pronounced, but it was not

statistically significant. Next, we conducted beta

diversity analysis, which was used to compare

microbial community compositions and assess

differences between microbial communities (Figure

8C). The principal coordinates analysis (PCoA) plot

showed a difference in the microbial communities

among the control, CCl4 and UA groups. Interestingly,

there was a slight difference in the microbial

communities among the NOX4-/-+UA group, the

RhoAi+UA group and the UA group. The composition

of the intestinal microbiota was also found to change

(Figure 8D). At the phylum level, the operational

taxonomic units (OTUs) of the beneficial bacteria

Firmicutes were lower in the CCl4-treated mice than in

the control mice. However, after UA treatment, this

decrease in the abundance of Firmicutes bacteria was

reversed. Firmicutes are beneficial bacteria that protect

the body through a variety of mechanisms [30–32]. The

abundance of Verrucomicrobia showed the opposite

trend. UA also improved microbial abundance at the

genus level (P < 0.05). At the genus level, the

abundances of Bacteroidales and Lachnospiraceae in

the CCl4 group were lower than those in the control

group. In the UA group, the abundances of both

Bacteroidales and Lachnospiraceae increased (Figure

8E). Furthermore, linear discriminant analysis effect

size (LEfSe) was used to analyse the microbiota

composition (Figure 8F–8G) and revealed the micro-

biota abundance within each group.

DISCUSSION

In this study, we show that the traditional Chinese

medicine UA can alleviate CCl4-induced liver fibrosis.

We explored the underlying mechanisms involved in

this effect and found that the UA-induced reversal of

liver fibrosis may be achieved by inhibiting the

NOX4/ROS and RhoA/ROCK signalling pathways,

which may interact with each other. The conversion of

HSCs to proliferating MFBs, which release ECM that is

then deposited, is a central event in the pathogenesis of

hepatic fibrosis [33, 34]. NOX has been shown to

convert catalytic oxygen molecules to ROS, which in

turn regulates the activation of HSCs [35–37].

Furthermore, NOX4 can mediate TGF-β and other

signalling pathways and induce the activation of resting

HSCs and apoptosis in hepatocytes [38, 39]; NOX4

therefore plays an important role in the progression of

liver fibrosis.


Figure 7. Interaction between NOX4/ROS and RhoA/ROCK1 in liver fibrotic mice. (A) In liver fibrotic mice in which NOX4 expression was inhibited, RhoA/ROCK1 expression was detected by IHC. (B) Dual immunofluorescence staining of liver sections from mice in the control, CCl4, NOX4-/- and AP groups stained for nuclei (DAPI, blue), aHSCs (α-SMA, green), and RhoA (red), and the merged images are shown. (C) Hepatic mRNA levels of RhoA/ROCK1 were measured by qRT-PCR. (D) In liver fibrotic mice in which RhoA expression was inhibited, NOX4 expression was detected by IHC. (E) Detection of the MDA content in the liver by a commercial kit. (F) Hepatic mRNA levels of NOX4 were measured by qRT-PCR. Data represent the mean ± SD of each group. *P < 0.05 and ***P < 0.001.


Figure 8. Effect of UA on intestinal microbiota dysbiosis in mice with CCl4-induced liver fibrosis. (A) Ileal mRNA levels of the TJ proteins ZO-1 and occludin were measured by qRT-PCR. (B) The alpha diversity of each group was assessed by determining the Shannon index and the Chao1 index. (C) PCoA to determine the weighted UniFrac distance of the intestinal microbiota. (D) Composition of the intestinal microbiota of each group at the phylum level. (E) Composition of the intestinal microbiota of each group at the genus level. (F) Linear discriminant analysis effect size (LEfSe) prediction was used to identify the bacteria in each group with the most differential abundance. (G) Linear discriminant analysis (LDA) scores showed significant differences in the bacteria in each group. Only the bacteria whose abundance met an LDA threshold value of >2 are shown. Data represent the mean ± SD of each group. *P < 0.05 and ***P < 0.001.


In previous experiments, we induced NOX4 activation

in HSCs and found that the intracellular ROS levels,

proliferation, migration, the degree of cytoskeleton F-

actin polymerization and profibrotic factor expression

decreased. These results were also confirmed with

animal experiments. Injection of NOX4 bioinhibitors

into CCl4-induced liver fibrosis in mice reversed the

progression of liver fibrosis [40]. Here, we used NOX4

knockout mice to better understand the role of NOX4 in

the development of liver fibrosis in vivo. Pathological

analysis of mouse liver tissue showed that liver fibrosis

and inflammatory infiltration in the NOX4-/- group were

significantly reduced compared with those in the CCl4

group. In NOX4 knockout liver fibrotic mice, the levels

of serum indicators that reflect the expression of the

ROS indicator MDA and the expression of liver

fibrosis-related factors also declined to a certain degree,

indicating that the NOX4/ROS signalling pathway plays

a role in improving liver fibrosis and that intervention in

this pathway can reverse the progression of liver

fibrosis.

We also demonstrated in vitro that after induction of

RhoA activation in HSCs, cellular proliferation and

migration and the degree of cytoskeletal F-actin

polymerization increased, and the expression of liver

fibrosis-related factors also increased. To demonstrate

the role of RhoA in liver fibrosis in vivo, we

constructed liver fibrotic mice in which RhoA was

inhibited with AAV viruses or inhibitors. Pathological

analysis of mouse liver tissue showed that liver fibrosis

and inflammatory infiltration in the RhoAi group and

FA group were significantly reduced compared with

those in the CCl4 group. In RhoA-inhibited liver fibrotic

mice, the levels of serum indicators and the expression

of liver fibrosis-related factors also declined to a certain

degree, indicating that the RhoA/ROCK1 signalling

pathway plays a role in improving liver fibrosis and that

intervention with RhoA can alleviate the progression of

liver fibrosis.

The relationship between NOX4/ROS and RhoA/

ROCK1, two signalling pathways that play a role in

fibrotic diseases, is not clearly defined. In pulmonary

fibrosis, NOX4/ROS is an upstream signalling pathway

of RhoA/ROCK1 [11]. Interestingly, in renal fibrosis,

RhoA/ROCK1 aggravates renal fibrosis by activating

the NOX4/ROS signalling pathway [12].

We aimed to determine the relationship between

NOX4/ROS and RhoA/ROCK1 in liver fibrosis. We

found through previous in vitro experiments that high

expression levels of NOX4, but not RhoA, can increase

the level of ROS in HSCs. Furthermore, the expression

of RhoA was decreased after the inhibition of NOX4

expression. The inhibition of RhoA expression did not

affect the expression of NOX4. This result suggests that

NOX4 activates HSCs by upregulating the expression

of RhoA. In this experiment, we again assessed the

relationship between NOX4 and RhoA in an animal

model. In the livers of NOX4 knockout liver fibrotic

mice, the expression of RhoA and ROCK1 decreased,

while in RhoA-inhibited liver fibrotic mice, the

expression of NOX4 did not show any obvious change.

This suggests that NOX4/ROS may be the upstream

signalling pathway of RhoA/ROCK1 in liver fibrosis. In

addition, RhoA-related signalling pathways have been

shown to be closely linked to human liver cirrhosis and

related complications such as portal hypertension [24].

Clarifying the relationship between NOX4/ROS and

RhoA/ROCK1 can help elucidate the progression of

chronic liver disease.

As a traditional Chinese medicine, UA can inhibit the

proliferation of activated HSCs, induce apoptosis, and

protect liver cells to exert anti-fibrotic effects [41]. We

conducted a preliminary exploration of the target of UA

in HSCs and found that UA can inhibit the proliferation

and migration of HSCs by inhibiting the NOX4/ROS

and RhoA/ROCK1 signalling pathways, and this result

was further verified in vivo. In liver fibrotic mice in

which NOX4 and RhoA were inhibited, the effect of

UA in improving liver fibrosis declined to a certain

degree, and the expression of liver fibrosis-related

factors was also altered. This suggests that NOX4 and

RhoA are two important targets by which UA exerts its

anti-fibrotic effects. Unfortunately, limited by objective

factors, we have not performed NOX4 and RhoA

recovery experiments in animals to verify the

relationship between NOX4/ROS and RhoA/ROCK1.

More animal models are needed to verify the interaction

between NOX4/ROS and RhoA/ROCK1.

Another important finding of this study is the change in

intestinal microbiota during UA treatment of liver

fibrosis. As adjacent organs, the liver and intestine are

closely connected. The two interact and influence each

other, and this association is referred to as the "liver-gut

axis". Therefore, disorders of the normal metabolic

activities of the liver in chronic liver disease cause

damage to the intestinal environment through the liver-

gut axis [42, 43]. In liver cirrhosis, the intestinal

microbiota becomes disordered, which may be due to a

reduction in bile acid secretion and changes to the

intestinal microbiota composition in cirrhosis, and the

excretion of factors in the intestine affects the intestinal

microbiota [44]. However, studies on intestinal

microbiota changes in liver fibrosis are rare. To this end,

we explored changes to the intestinal microbiota during

liver fibrosis and its improvement following treatment

with UA. In the liver fibrosis animal model, the diversity

and abundance of the microbiota were significantly


decreased. In terms of the microbiota composition, liver

fibrotic mice also have fewer beneficial bacteria than

normal mice, indicating a disorder in the intestinal

microbiota. Furthermore, the disordered microbiota is

likely to stimulate LPS-induced Toll-like receptor

(TLR)-related pathway activation through the damaged

intestinal barrier, aggravating intrahepatic inflammation

and even liver fibrosis [45, 46]. In contrast, when liver

fibrotic mice were treated with UA, the decreased

diversity and abundance of the intestinal microbiota

showed a significant increase, and beneficial bacteria

also showed obvious recovery, suggesting that UA

improves the intestinal microbiota. Although the current

mechanism by which UA improves intestinal microbiota

disorders is not clear, this study found that this

phenomenon may be related to the inhibition of NOX4

and RhoA by UA-based microbiota changes in the

NOX4 intervention and RhoA intervention groups.

However, it is unknown whether UA directly affects the

intestinal microbiota by inhibiting NOX4 and RhoA or

indirectly improves the intestinal microbiota by

reducing liver fibrosis. In addition, some researchers

have recently attempted to develop characteristic

microbiota as a non-invasive biomarker and a micro-

biota signature for the classification, diagnosis, and

treatment of chronic live diseases [47, 48]. Based on the

results of this study, we can look for the microbial

signature of liver fibrosis to better diagnose liver

fibrosis and evaluate the efficacy of UA in clinical.

More evidence is required to demonstrate the potential

mechanisms of UA in the improvement of the intestinal

microbiota, and more sophisticated experiments and

models will need to be conducted in the future.

In conclusion, our results indicate that NOX4/ROS and

RhoA/ROCK1, which may interact, play an important

role in the development of liver fibrosis. Furthermore,

UA may reverse liver fibrosis by intervening in these

two signalling pathways. The potential mechanism of the

anti-fibrotic effects of UA is still being explored. We

aimed to clarify the possible molecular targets of UA

and provide reasonable experimental evidence for the

future use of UA in clinical treatment. These findings

provide new insight into UA treatment of liver fibrosis.

However, further reasonable in vivo and in vitro

experiments are needed to confirm these results.

Combined with our previous research results, the

results described in this paper provide a reasonable

experimental basis for the clinical application of UA.

MATERIALS AND METHODS

Experimental animal model and design

The wild-type (WT) C57BL/6 mice used in the

experiments were from the Department of Laboratory

Animal Science of Nanchang University, and NOX4

knockout C57BL/6 mice were purchased from the

Jackson Laboratory (USA). All animals were maintained

in a temperature-controlled environment (20–22 °C)

with a 12 h light-dark cycle with free access to sterile

food and water. Based on widely recognized research,

carbon tetrachloride (CCl4) was selected to induce liver

fibrosis in mice [17, 18]. According to the principle of

random allocation, C57BL/6 mice weighing 20 to 30 g

were randomly divided into the control group [n = 8,

gavage with olive oil (2 ml/kg) twice a week for 8

weeks], CCl4 group [n = 8, gavage with CCl4 at 2 ml/kg

(20% olive oil dilution) twice a week for 8 weeks], and

UA group [n = 8, mice that were administered CCl4 for 4

weeks received continued CCl4 and UA (40 mg/kg/day)

gavage for 4 weeks]. NOX4 knockout mice were

randomly divided into the NOX4-/- group (n = 8, gavage

with CCl4 twice a week for 8 weeks) and the NOX4-/- +

UA group (n = 8, treatment consistent with that of the

UA group). A group of C57BL/6 mice that received

adeno-associated virus (AAV) (4 ml/kg) via tail vein

injection for 1 week to inhibit RhoA were randomly

divided into the RhoAi group (n = 8, gavage with olive

oil twice a week for 8 weeks) and the RhoAi + UA

group (n = 8, treatment consistent with that of the UA

group). We also added 2 inhibitor groups: the apocynin

(AP) group [n = 8, mice that were given CCl4 by gavage

for 4 weeks and CCl4 plus AP (40 mg/kg/d) (NOX4

biological inhibitor) by gavage for 4 weeks] and the

fasudil (FA) group [n = 8, mice received CCl4 by gavage

for 4 weeks and CCl4 plus FA (10 mg/kg/d) (RhoA

biological inhibitor) by gavage for 4 weeks]. The mice

were subjected to experimental procedures approved by

the Animal Care and Use Committee of Nanchang

University. All procedures were performed according to

the National Institutes of Health Guide for the Care and

Use of Laboratory Animals.

Measurement of blood indices

Serum samples were collected while sacrificing the

mice, and the alanine aminotransferase (ALT), aspartate

aminotransferase (AST), and total bilirubin (TBIL)

levels were measured by using an automatic biochemical

analyser (Department of Clinical Laboratory, First

Affiliated Hospital of Nanchang University, China).

Histological analysis

The liver samples were fixed in 4% neutral-buffered

formalin, embedded in paraffin and sectioned. Sections

underwent haematoxylin and eosin (HE) staining,

Masson’s trichrome staining, immunohistochemistry

(IHC), TdT-mediated dUTP nick-end labelling

(TUNEL) analysis, and immunofluorescent analysis and

were evaluated by microscopy. We randomly selected


five visual fields for observation, scored liver fibrosis

using METAVIR scoring criteria, and evaluated

intestinal mucosal damage using the Chiu scoring

method. IHC was used to determine the localization and

expression of related proteins. Liver fibrosis was

estimated by Masson’s trichrome staining. Specimens

were incubated with an appropriate antibody and were

observed and photographed by confocal microscopy.

Hydroxyproline and malondialdehyde measurements

The hydroxyproline and malondialdehyde (MDA)

contents of the liver tissues were determined using an

assay kit from Nan-jing Jiancheng Bioengineering

Institute (Nanjing, China) according to the manufac-

turer’s instructions.

Construction and injection of vectors for RhoA

inhibition

We constructed an AAV for RhoA inhibition. AAVs

carrying short hairpin RNA (shRNA) using CV266 as a

vector were injected into the mouse via the tail vein. The

final titre of AAV-shRhoA was 3.5 × 1012 viral

particles/ml.

Gut microbiota analysis

We used a stool DNA kit (Omega, China) to extract

genomic DNA from the faeces collected while

sacrificing mice, and then purity was verified by 1%

agarose gel electrophoresis (BioFROXX, China).

Primers (338F 5'-ACTCCTACGGGAGGCAGCAG-3'

and 806R 5'-GGACTACHVGGGTWTCTAAT-3')

were used to amplify the bacterial V3-V4 region of the

16S rRNA gene. Pyrosequencing of the PCR products

was performed on an Illumina MiSeq Instrument.

(Majorbio, China). Analysis of changes in intestinal

microbiota by alpha diversity (Shannon and Chao1

index), beta diversity, and community composition

were calculated.

Western blotting analysis

Liver tissue protein was obtained from tissue lysates for

western blotting. The protein levels were determined

using a BCA assay kit (Tiangen, Beijing, China).

Denatured proteins were separated on 10% Tris-glycine

polyacrylamide gels by SDS-PAGE and transferred to

PVDF membranes. The PVDF membranes were

incubated overnight at 4 °C with anti-collagen I (Abcam,

Cat. ab138492), anti-MMP1 (Abcam, Cat. ab137332),

anti-α -SMA (Abcam, Cat. ab32575), anti-TIMP1

(Abcam, Cat. ab109125), anti-NOX4 (Abcam, Cat.

ab109225), anti-RhoA (Abcam, Cat. ab187027), anti-

ROCK1 (Abcam, Cat. ab45171), and anti-GAPDH

(Abcam, Cat. ab8245). The corresponding membrane-

bound antibodies were detected by a hypersensitive

chemiluminescence detection reagent.

Quantitative real-time RT-PCR (qRT-PCR)

For real-time PCR analysis, PCR was performed with

a reaction mixture containing cDNA template,

primers, and TB Green™ Fast qPCR Mix (TaKaRa)

in a Step One Plus Real-Time PCR System (Thermo

Fisher Scientific). The relative abundances of the

target genes were obtained by comparison against a

standard curve.

Statistical analysis

We used SPSS 23.0 software for data analysis. Image

production and output were performed using

GraphPad Prism 7.0 software. Each experiment was

repeated 3 times to ensure confidence in the results.

One-way analysis of variance (ANOVA), Student’s t

test, Mann-Whitney rank sum test, or Kruskal-Wallis

H test was used to analyse the significant differences

between groups. P <0.05 was considered significant.

Abbreviations

ECM: extracellular matrix; UA: ursolic acid; AP:

apocynin; FA: fasudil; HSCs: hepatic stellate cells;

NOX: NADPH oxidase; ROS: reactive oxygen species;

MFBs: myofibroblasts; CCl4: carbon tetrachloride;

AAV: adeno-associated virus; ALT: aminotransferase;

AST: aspartate aminotransferase; TBIL: total bilirubin;

MDA: malondialdehyde.

AUTHOR CONTRIBUTIONS

SW and FL contributed equally to this study. SW and FL

designed and wrote the manuscript. CH, CL, and QL

analysed the data. XZ critically revised the manuscript.

ACKNOWLEDGMENTS

We would like to thank the National Natural Science

Foundation of China for the economic support.

CONFLICTS OF INTEREST

The authors declare that there are no conflicts of

interest.

FUNDING

This study was supported by the National Natural Science

Foundation of China (grant number: 81960120) and the

“Gan-Po Talent 555” Project of Jiangxi Province.


REFERENCES

1. Thompson AI, Conroy KP, Henderson NC. Hepatic stellate cells: central modulators of hepatic carcinogenesis. BMC Gastroenterol. 2015; 15:63.

https://doi.org/10.1186/s12876-015-0291-5 PMID:26013123

2. Schuppan D, Kim YO. Evolving therapies for liver fibrosis. J Clin Invest. 2013; 123:1887–901.

https://doi.org/10.1172/JCI66028 PMID:23635787

3. Zhang DY, Friedman SL. Fibrosis-dependent mechanisms of hepatocarcinogenesis. Hepatology. 2012; 56:769–75.

https://doi.org/10.1002/hep.25670 PMID:22378017

4. Lozano R, Naghavi M, Foreman K, Lim S, Shibuya K, Aboyans V, Abraham J, Adair T, Aggarwal R, Ahn SY, Alvarado M, Anderson HR, Anderson LM, et al. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the global burden of disease study 2010. Lancet. 2012; 380:2095–128.

https://doi.org/10.1016/S0140-6736(12)61728-0 PMID:23245604

5. Tsochatzis EA, Bosch J, Burroughs AK. Liver cirrhosis. Lancet. 2014; 383:1749–61.

https://doi.org/10.1016/S0140-6736(14)60121-5 PMID:24480518

6. Kim KS, Hur W, Park SJ, Hong SW, Choi JE, Goh EJ, Yoon SK, Hahn SK. Bioimaging for targeted delivery of hyaluronic acid derivatives to the livers in cirrhotic mice using quantum dots. ACS Nano. 2010; 4:3005–14.

https://doi.org/10.1021/nn100589y PMID:20518553

7. Paik YH, Iwaisako K, Seki E, Inokuchi S, Schnabl B, Osterreicher CH, Kisseleva T, Brenner DA. The nicotinamide adenine dinucleotide phosphate oxidase (NOX) homologues NOX1 and NOX2/gp91(phox) mediate hepatic fibrosis in mice. Hepatology. 2011; 53:1730–41.


8. Porter AP, Papaioannou A, Malliri A. Deregulation of rho GTPases in cancer. Small GTPases. 2016; 7:123–38.

https://doi.org/10.1080/21541248.2016.1173767 PMID:27104658

9. Li L, Wang JY, Yang CQ, Jiang W. Effect of RhoA on transforming growth factor β1-induced rat hepatic stellate cell migration. Liver Int. 2012; 32:1093–102.

https://doi.org/10.1111/j.1478-3231.2012.02809.x PMID:22498718

10. Ridley AJ. Historical overview of rho GTPases. Methods Mol Biol. 2012; 827:3–12.

https://doi.org/10.1007/978-1-61779-442-1_1 PMID:22144264

11. Meng Y, Li T, Zhou GS, Chen Y, Yu CH, Pang MX, Li W, Li Y, Zhang WY, Li X. The angiotensin-converting enzyme 2/angiotensin (1-7)/mas axis protects against lung fibroblast migration and lung fibrosis by inhibiting the NOX4-derived ROS-mediated RhoA/rho kinase pathway. Antioxid Redox Signal. 2015; 22:241–58.

https://doi.org/10.1089/ars.2013.5818 PMID:25089563

12. Manickam N, Patel M, Griendling KK, Gorin Y, Barnes JL. RhoA/rho kinase mediates TGF-β1-induced kidney myofibroblast activation through Poldip2/Nox4-derived reactive oxygen species. Am J Physiol Renal Physiol. 2014; 307:F159–71.

https://doi.org/10.1152/ajprenal.00546.2013 PMID:24872317

13. Paik YH, Kim J, Aoyama T, De Minicis S, Bataller R, Brenner DA. Role of NADPH oxidases in liver fibrosis. Antioxid Redox Signal. 2014; 20:2854–72.

https://doi.org/10.1089/ars.2013.5619 PMID:24040957

14. Ikeda Y, Murakami A, Fujimura Y, Tachibana H, Yamada K, Masuda D, Hirano K, Yamashita S, Ohigashi H. Aggregated ursolic acid, a natural triterpenoid, induces IL-1beta release from murine peritoneal macrophages: role of CD36. J Immunol. 2007; 178:4854–64.

https://doi.org/10.4049/jimmunol.178.8.4854 PMID:17404266

15. Cargnin ST, Gnoatto SB. Ursolic acid from apple pomace and traditional plants: a valuable triterpenoid with functional properties. Food Chem. 2017; 220:477–89.

https://doi.org/10.1016/j.foodchem.2016.10.029 PMID:27855928

16. Wang X, Ikejima K, Kon K, Arai K, Aoyama T, Okumura K, Abe W, Sato N, Watanabe S. Ursolic acid ameliorates hepatic fibrosis in the rat by specific induction of apoptosis in hepatic stellate cells. J Hepatol. 2011; 55:379–87.

https://doi.org/10.1016/j.jhep.2010.10.040 PMID:21168456

17. Pierce RA, Glaug MR, Greco RS, Mackenzie JW, Boyd CD, Deak SB. Increased procollagen mRNA levels in carbon tetrachloride-induced liver fibrosis in rats. J Biol Chem. 1987; 262:1652–58.

PMID:3805048

18. Pérez Tamayo R. Is cirrhosis of the liver experimentally produced by CCl4 and adequate model of human cirrhosis? Hepatology. 1983;

https://doi.org/10.1186/s12876-015-0291-5

https://pubmed.ncbi.nlm.nih.gov/26013123

https://doi.org/10.1172/JCI66028


https://doi.org/10.1002/hep.25670


https://doi.org/10.1016/S0140-6736(12)61728-0


https://doi.org/10.1016/S0140-6736(14)60121-5


https://doi.org/10.1021/nn100589y




https://doi.org/10.1080/21541248.2016.1173767


https://doi.org/10.1111/j.1478-3231.2012.02809.x


https://doi.org/10.1007/978-1-61779-442-1_1


https://doi.org/10.1089/ars.2013.5818


https://doi.org/10.1152/ajprenal.00546.2013


https://doi.org/10.1089/ars.2013.5619


https://doi.org/10.4049/jimmunol.178.8.4854


https://doi.org/10.1016/j.foodchem.2016.10.029


https://doi.org/10.1016/j.jhep.2010.10.040





19. Lee YA, Wallace MC, Friedman SL. Pathobiology of liver fibrosis: a translational success story. Gut. 2015; 64:830–41.

https://doi.org/10.1136/gutjnl-2014-306842 PMID:25681399

20. Iredale JP, Benyon RC, Pickering J, McCullen M, Northrop M, Pawley S, Hovell C, Arthur MJ. Mechanisms of spontaneous resolution of rat liver fibrosis. Hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors. J Clin Invest. 1998; 102:538–49.

https://doi.org/10.1172/JCI1018 PMID:9691091

21. Leroy V, Monier F, Bottari S, Trocme C, Sturm N, Hilleret MN, Morel F, Zarski JP. Circulating matrix metalloproteinases 1, 2, 9 and their inhibitors TIMP-1 and TIMP-2 as serum markers of liver fibrosis in patients with chronic hepatitis C: comparison with PIIINP and hyaluronic acid. Am J Gastroenterol. 2004; 99:271–79.


22. Liang S, Kisseleva T, Brenner DA. The role of NADPH oxidases (NOXs) in liver fibrosis and the activation of myofibroblasts. Front Physiol. 2016; 7:17.

https://doi.org/10.3389/fphys.2016.00017 PMID:26869935

23. Ni J, Dong Z, Han W, Kondrikov D, Su Y. The role of RhoA and cytoskeleton in myofibroblast transformation in hyperoxic lung fibrosis. Free Radic Biol Med. 2013; 61:26–39.

https://doi.org/10.1016/j.freeradbiomed.2013.03.012 PMID:23517783

24. Klein S, Rick J, Lehmann J, Schierwagen R, Schierwagen IG, Verbeke L, Hittatiya K, Uschner FE, Manekeller S, Strassburg CP, Wagner KU, Sayeski PP, Wolf D, et al. Janus-kinase-2 relates directly to portal hypertension and to complications in rodent and human cirrhosis. Gut. 2017; 66:145–55.


25. Kalaitzakis E. Gastrointestinal dysfunction in liver cirrhosis. World J Gastroenterol. 2014; 20:14686–95.

https://doi.org/10.3748/wjg.v20.i40.14686 PMID:25356031

26. Palma P, Mihaljevic N, Hasenberg T, Keese M, Koeppel TA. Intestinal barrier dysfunction in developing liver cirrhosis: an in vivo analysis of bacterial translocation. Hepatol Res. 2007; 37:6–12.

https://doi.org/10.1111/j.1872-034X.2007.00004.x PMID:17300693

27. Assimakopoulos SF, Tsamandas AC, Tsiaoussis GI, Karatza E, Triantos C, Vagianos CE, Spiliopoulou I, Kaltezioti V, Charonis A, Nikolopoulou VN, Scopa CD, Thomopoulos KC. Altered intestinal tight junctions’ expression in patients with liver cirrhosis: a pathogenetic mechanism of intestinal hyperpermeability. Eur J Clin Invest. 2012; 42:439–46.


28. Odenwald MA, Turner JR. The intestinal epithelial barrier: a therapeutic target? Nat Rev Gastroenterol Hepatol. 2017; 14:9–21.

https://doi.org/10.1038/nrgastro.2016.169 PMID:27848962

29. Schnabl B, Brenner DA. Interactions between the intestinal microbiome and liver diseases. Gastroenterology. 2014; 146:1513–24.

https://doi.org/10.1053/j.gastro.2014.01.020 PMID:24440671

30. Caminero A, McCarville JL, Zevallos VF, Pigrau M, Yu XB, Jury J, Galipeau HJ, Clarizio AV, Casqueiro J, Murray JA, Collins SM, Alaedini A, Bercik P, et al. Lactobacilli degrade wheat amylase trypsin inhibitors to reduce intestinal dysfunction induced by immunogenic wheat proteins. Gastroenterology. 2019; 156:2266–80.

https://doi.org/10.1053/j.gastro.2019.02.028 PMID:30802444

31. Malik M, Suboc TM, Tyagi S, Salzman N, Wang J, Ying R, Tanner MJ, Kakarla M, Baker JE, Widlansky ME. Lactobacillus plantarum 299v supplementation improves vascular endothelial function and reduces inflammatory biomarkers in men with stable coronary artery disease. Circ Res. 2018; 123:1091–102.

https://doi.org/10.1161/CIRCRESAHA.118.313565 PMID:30355158

32. Riehl TE, Alvarado D, Ee X, Zuckerman A, Foster L, Kapoor V, Thotala D, Ciorba MA, Stenson WF. Lactobacillus rhamnosus GG protects the intestinal epithelium from radiation injury through release of lipoteichoic acid, macrophage activation and the migration of mesenchymal stem cells. Gut. 2019; 68:1003–13.


33. Oh Y, Park O, Swierczewska M, Hamilton JP, Park JS, Kim TH, Lim SM, Eom H, Jo DG, Lee CE, Kechrid R, Mastorakos P, Zhang C, et al. Systemic PEGylated TRAIL treatment ameliorates liver cirrhosis in rats by eliminating activated hepatic stellate cells. Hepatology. 2016; 64:209–23.

https://doi.org/10.1002/hep.1840030118


https://doi.org/10.1136/gutjnl-2014-306842


https://doi.org/10.1172/JCI1018


https://doi.org/10.1111/j.1572-0241.2004.04055.x


https://doi.org/10.3389/fphys.2016.00017


https://doi.org/10.1016/j.freeradbiomed.2013.03.012




https://doi.org/10.3748/wjg.v20.i40.14686


https://doi.org/10.1111/j.1872-034X.2007.00004.x


https://doi.org/10.1111/j.1365-2362.2011.02609.x


https://doi.org/10.1038/nrgastro.2016.169


https://doi.org/10.1053/j.gastro.2014.01.020


https://doi.org/10.1053/j.gastro.2019.02.028


https://doi.org/10.1161/CIRCRESAHA.118.313565






34. Lafdil F, Chobert MN, Couchie D, Brouillet A, Zafrani ES, Mavier P, Laperche Y. Induction of Gas6 protein in CCl4-induced rat liver injury and anti-apoptotic effect on hepatic stellate cells. Hepatology. 2006; 44:228–39.


35. Ceci R, Duranti G, Leonetti A, Pietropaoli S, Spinozzi F, Marcocci L, Amendola R, Cecconi F, Sabatini S, Mariottini P, Cervelli M. Adaptive responses of heart and skeletal muscle to spermine oxidase overexpression: evaluation of a new transgenic mouse model. Free Radic Biol Med. 2017; 103:216–25.


36. Chang KH, Park JM, Lee CH, Kim B, Choi KC, Choi SJ, Lee K, Lee MY. NADPH oxidase (NOX) 1 mediates cigarette smoke-induced superoxide generation in rat vascular smooth muscle cells. Toxicol In Vitro. 2017; 38:49–58.

https://doi.org/10.1016/j.tiv.2016.10.013 PMID:27816504

37. Kleinschmidt EG, Schlaepfer DD. Focal adhesion kinase signaling in unexpected places. Curr Opin Cell Biol. 2017; 45:24–30.

https://doi.org/10.1016/j.ceb.2017.01.003 PMID:28213315

38. Aoyama T, Paik YH, Watanabe S, Laleu B, Gaggini F, Fioraso-Cartier L, Molango S, Heitz F, Merlot C, Szyndralewiez C, Page P, Brenner DA. Nicotinamide adenine dinucleotide phosphate oxidase in experimental liver fibrosis: GKT137831 as a novel potential therapeutic agent. Hepatology. 2012; 56:2316–27.


39. Jiang JX, Chen X, Serizawa N, Szyndralewiez C, Page P, Schröder K, Brandes RP, Devaraj S, Török NJ. Liver fibrosis and hepatocyte apoptosis are attenuated by GKT137831, a novel NOX4/NOX1 inhibitor in vivo. Free Radic Biol Med. 2012; 53:289–96.


40. Gan D, Zhang W, Huang C, Chen J, He W, Wang A, Li B, Zhu X. Ursolic acid ameliorates CCl4-induced liver fibrosis through the NOXs/ROS pathway. J Cell Physiol. 2018; 233:6799–813.

https://doi.org/10.1002/jcp.26541 PMID:29672850

41. He W, Shi F, Zhou ZW, Li B, Zhang K, Zhang X, Ouyang C, Zhou SF, Zhu X. A bioinformatic and mechanistic study elicits the antifibrotic effect of ursolic acid

through the attenuation of oxidative stress with the involvement of ERK, PI3K/akt, and p38 MAPK signaling pathways in human hepatic stellate cells and rat liver. Drug Des Devel Ther. 2015; 9:3989–4104.

https://doi.org/10.2147/DDDT.S85426 PMID:26347199

42. Liu HX, Keane R, Sheng L, Wan YJ. Implications of microbiota and bile acid in liver injury and regeneration. J Hepatol. 2015; 63:1502–10.


43. Wiest R, Albillos A, Trauner M, Bajaj JS, Jalan R. Targeting the gut-liver axis in liver disease. J Hepatol. 2017; 67:1084–103.


44. Lorenzo-Zúñiga V, Bartolí R, Planas R, Hofmann AF, Viñado B, Hagey LR, Hernández JM, Mañé J, Alvarez MA, Ausina V, Gassull MA. Oral bile acids reduce bacterial overgrowth, bacterial translocation, and endotoxemia in cirrhotic rats. Hepatology. 2003; 37:551–57.

https://doi.org/10.1053/jhep.2003.50116 PMID:12601352

45. Soares JB, Pimentel-Nunes P, Roncon-Albuquerque R, Leite-Moreira A. The role of lipopolysaccharide/toll-like receptor 4 signaling in chronic liver diseases. Hepatol Int. 2010; 4:659–72.

https://doi.org/10.1007/s12072-010-9219-x PMID:21286336

46. Zhu Q, Zou L, Jagavelu K, Simonetto DA, Huebert RC, Jiang ZD, DuPont HL, Shah VH. Intestinal decontamination inhibits TLR4 dependent fibronectin-mediated cross-talk between stellate cells and endothelial cells in liver fibrosis in mice. J Hepatol. 2012; 56:893–99.


47. Dong TS, Katzka W, Lagishetty V, Luu K, Hauer M, Pisegna J, Jacobs JP. A microbial signature identifies advanced fibrosis in patients with chronic liver disease mainly due to NAFLD. Sci Rep. 2020; 10:2771.

https://doi.org/10.1038/s41598-020-59535-w PMID:32066758

48. Caussy C, Tripathi A, Humphrey G, Bassirian S, Singh S, Faulkner C, Bettencourt R, Rizo E, Richards L, Xu ZZ, Downes MR, Evans RM, Brenner DA, et al. A gut microbiome signature for cirrhosis due to nonalcoholic fatty liver disease. Nat Commun. 2019; 10:1406.

https://doi.org/10.1038/s41467-019-09455-9 PMID:30926798







https://doi.org/10.1016/j.tiv.2016.10.013


https://doi.org/10.1016/j.ceb.2017.01.003






https://doi.org/10.1002/jcp.26541


https://doi.org/10.2147/DDDT.S85426






https://doi.org/10.1053/jhep.2003.50116


https://doi.org/10.1007/s12072-010-9219-x




https://doi.org/10.1038/s41598-020-59535-w


https://doi.org/10.1038/s41467-019-09455-9



SUPPLEMENTARY MATERIALS

Supplementary Figures

Supplementary Figure 1. NOX4-/- mouse genotype identification. (A) NOX4 DNA was validated by agarose gel electrophoresis. WT: wild-type mice; F1: B6.129-NOX4tm1Kkr/J mouse progeny; vehicle: DNA was replaced with water, negative control group.


Supplementary Figure 2. Validation of RhoA expression inhibition in the mouse model. (A) Hepatic mRNA levels of RhoA were measured by qRT-PCR. (B) RhoA protein expression was detected by a western blot. (C) Histogram analysis of the levels of RhoA. Data represent the mean ± SD of each group. *P < 0.05 and ***P < 0.001.

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Research Paper Ursolic acid reverses liver fibrosis by …...Ursolic acid reverses liver fibrosis by inhibiting interactive NOX4/ROS and RhoA/ROCK1 signalling pathways Sizhe Wan1,*,